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dc.contributor.advisorO'Hagan, David
dc.contributor.authorThompson, Stephen
dc.coverage.spatial326en_US
dc.date.accessioned2015-11-17T12:48:10Z
dc.date.available2015-11-17T12:48:10Z
dc.date.issued2015-11-30
dc.identifier.urihttps://hdl.handle.net/10023/7805
dc.description.abstractThe fluorinase enzyme, originally isolated from Streptomyces cattleya, has the unique ability to generate a C–F bond from aqueous fluoride ion and S-adenosylmethionine, making the fluorinase an attractive biochemical tool for radiolabelling biomolecules with fluorine-18 for application to positron emission tomography (PET). The inherent substrate specificity of the enzyme is, however, limiting, as only small modifications to the natural nucleoside substrate were known to be tolerated. This thesis describes an exploration and expansion of the substrate scope of the fluorinase enzyme, and its application to radiolabelling biomolecules for PET. The design and synthesis of a novel acetylene bearing substrate for the fluorinase, 5'-chloro-5'-deoxy-2-ethynyladenosine (ClDEA) is described. ClDEA proved an excellent substrate for the fluorinase, and the kinetics of the transformation and binding affinities of the new substrate and product were investigated. The fluorinated acetylenic product was demonstrated to undergo a copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction with an azide bearing RGD peptide, and this methodology was investigated for the synthesis of a novel fluorine-18-bearing prosthetic group for the synthesis of a radiolabelled RGD peptide, which was assessed in vivo in a rat. After the demonstration that the fluorinase can be used for “last step” radiolabelling of bioactive peptides, the synthesis of dimeric and tetrameric RGD-bearing substrates for the fluorinase was investigated. These large constructs underwent efficient enzymatic fluorination, and the fluorinated products showed increased binding affinity to their targets, compared to monomeric analogues. The challenges encountered during radiolabelling of these multimers with fluorine-18 using the fluorinase are discussed. A difluoromethyl-bearing nucleoside substrate (F₂DA) was synthesised as a potential substrate in the reverse direction for the fluorinase, to further probe the substrate specificity if the fluorinase. Upon incubation with the enzyme, F₂DA did not appear to undergo reaction, despite the demonstration that F₂DA binds to the enzyme. Finally, the optimisation of a fluorinase-based protocol for the synthesis of the PET radiotracer [¹⁸F]fluoroacetate is described. The enzymatic method proved unsuitable for a small animal study due to contamination of the final product, and a chemical method was investigated and optimised as an alternative approach. [¹⁸F]Fluoroacetate synthesised using the developed chemical method was employed in an in vivo evaluation of acetyl CoA synthetase (ACSS2) activity in healthy and tumour-bearing mouse models, in an study to assess the activity of ACSS2 in breast and colon cancer models in mice.en_US
dc.language.isoenen_US
dc.publisherUniversity of St Andrews
dc.subjectPositron emission tomographyen_US
dc.subjectFluorinaseen_US
dc.subjectRadiolabellingen_US
dc.subjectSubstrate scopeen_US
dc.subject.lccQD412.F1T5
dc.subject.lcshEnzymesen_US
dc.subject.lcshOrganofluorine compoundsen_US
dc.subject.lcshTomography, Emissionen_US
dc.subject.lcshRadiolabelingen_US
dc.titleExploring the substrate scope of the fluorinase from Streptomyces cattleya for applications to positron emission tomographyen_US
dc.typeThesisen_US
dc.type.qualificationlevelDoctoralen_US
dc.type.qualificationnamePhD Doctor of Philosophyen_US
dc.publisher.institutionThe University of St Andrewsen_US


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